Use of Cunninghamella elegans lendner in methods for treating industrial wastewaters containing dyes

ABSTRACT

Use of a fungal biomass for treating industrial wastewaters containing at least one dye, wherein:
         i. the fungal biomass contains at least the fungal species  Cunninghamella elegans  Lendner;   ii. the fungal biomass absorbs the at least one dye, so as to obtain wastewater that is basically free of the at least one dye.

TECHNICAL FIELD OF THE INVENTION

The present invention relates in general to methods for treatingindustrial wastewaters containing dyes. More particularly, the presentinvention relates to the use of fungal species in methods for treatingindustrial wastewaters containing dyes.

TECHNICAL BACKGROUND OF THE INVENTION

Large amounts of dyes are used in various industrial fields, such asfood, drug, cosmetic, textile and tanning fields (McMullan et al.,2001). It is estimated that the annual world production of dyes is above700,000 tons, more than a half of which include dyes for textile fibers,15% are dyes for other substrates such as leather and paper, 25% areorganic pigments and the remaining portion is made up of dyes forparticular uses (McMullan et al., 2001, Pearce et al., 2003).

Depending on molecule charge, dyes can be classed into anionic (acid),cationic (basic) and non-ionic dyes. As an alternative, depending on thechromophore group they can be classed into azo, anthraquinone, indigo,stilbene dyes etc., or depending on their applications. Azo andanthraquinone dyes represent the two most widespread classes of dyes forindustrial applications (Soares et al., 2001). Azo dyes arecharacterized by the presence of a double bond N═N and by other groupsthat are hard to degrade (Martins et al., 2001) and represent more than50% of total production. Their fixing capacity is generally low and somore than 40% of the amount used gets into industrial waste, which has aclear color resulting therefrom, even after accurate purificationtreatments (O'Neill et al., 1999). Anthraquinone dyes represent thesecond class for industrial relevance and can be divided into dyesderived from indigo and from anthraquinone. They are prepared bysuccessive introduction of the substituents on the pre-formed skeletonof anthraquinone.

Every year 5% to 10% of the world production of textile dyes isdischarged into industrial wastewaters, which get in their turn intonatural waterways where they can cause great problems for theenvironment and for living organisms (Yesilada et al., 2003). As amatter of fact, conventional methods for treating wastewaters are notsufficient to completely remove most of the dyes, which therefore tendto accumulate in the environment due to their complex molecularstructure, designed on purpose for giving high stability to light, waterand oxidizing agents (Fu and Viraraghavan, 2002a).

Dyes are toxic substances as shown by ETAD (1989) in a test on animalsfor 4,000 dyes. They can also have a carcinogenic and mutagenic action,due to the formation of aromatic amines when they are degraded underanaerobiosis from bacteria, as was shown in several researches onfishes, mice and other animals (Weisburger et al., 2002). Genotoxic andcarcinogenic effects are also possible on men, on whom dyes cause atleast short-term phenomena of contact and inhaling irritation (Yesiladaet al., 2003).

When dyes get into surface water, indirect damages to ecosystems arelikewise serious. As a matter of fact, gas solubility is compromised andabove all water transparency properties are altered, which results inserious consequences for flora and fauna (Fu and Viraraghavan, 2002a).Lower penetration of sun rays causes indeed a reduction of oxygenconcentration, which can be in its turn fatal for most water organisms(Yesilada et al., 2003).

Toxic substances contained in waste of industries using dyes shouldtherefore be completely removed before being released into theenvironment (Knapp et al., 2001). Physical and chemical purificationmethods are not always applicable and/or effective and always involvehigh costs for firms (Fu and Viraraghavan, 2001, Robinson et al., 2001).

Chemical treatments exploiting oxidizing processes are among the mostused methods, above all thanks to their easy application. Some of them,however, involve the use of chemical compounds that are noxious formen's health and/or for the environment such as the use of bleachingagents (Knapp et al., 2001). Among the most widespread treatments thefollowing should be mentioned: treatment with H₂O₂ together with ironsalts, with sodium hypochlorite, with ozone, photochemical andphotocatalytic methods, electrochemical destruction (Robinson et al.,2001).

Physical methods based on the absorption of dyes into various abioticmatrices have proved to be effective in many cases. Decolourization byabsorption is mainly based on ion exchange, which is affected by severalfactors such as the interaction between the dye and the type ofsubstances used for absorption, temperature, pH, contact time, etc.Active carbons, peat, wood chips, filtration membranes are the most usedabsorbing agents. Absorption is often favored by the use of ultrasounds(Robinson et al., 2001, Crini, 2006).

A valid alternative to most traditional treatments of dyed wastewaters,characterized by low cost and low environmental impact, is the use ofbiologic systems, i.e. biomasses that are able to degrade toxicsubstances up to the mineralization thereof (biodegradation), or absorbthem more or less passively on their cell structures (biosortpion)(Banat et al., 1996).

Recently, several researches have shown that biosorption can be regardedas a valid alternative to chemical-physical methods and to microbialand/or enzymatic biodegradation. Such researches have pointed out thecapacity of various microbial biomasses (bacteria, yeasts, fungi andalgae) to absorb or accumulate dyes (Polman et al., 1996, Crini, 2006),and among the various types of biomass the fungal biomass has proved tobe particularly suitable, even if the mechanisms regulating absorptionhave not yet been fully explained (Knapp et al., 2001, Crini, 2006).

In studies on biosorption with fungal biomasses, Mitosporic fungi andZygomycetes, belonging to the genus Aspergillus, Penicillium,Myrothecium and Rhizopus, are mainly used. Only in some casesBasidiomycetes are used, since for these fungi the main decolourizationmechanism is degradation and, according to Knapp et al. (2001),absorption occurs only in the initial stage of fungus-dyes interaction,which allows to create a strong contact between chromophores anddegrading enzymes associated to the surface of hyphae.

Mechanisms regulating dye biosorption by the biomass seem to vary bothas a function of the chemical structure of the dye and as a function ofthe specific chemical and structural composition of the biomass used. Asa matter of fact, it was shown that some dyes have a particular affinityfor particular species of organisms (Robinson et al., 2001).

Fu and Viraraghavan (2002b), working with biomasses of Aspergillus nigerthat had been deactivated, dried, pulverized and subjected to variouschemical treatments, so as to selectively deactivate different chemicalgroups, have shown that dye biosorption preferably occurs on cell wall,where the main binding sites would be made up of amine and carboxylgroups. It should still be explained whether during biosorptionprocesses the dye is bound only to the outer surface or whether it canalso be carried, at least partially, into the hyphae (Polman andBreckenbridge, 1996; Brahimihorn et al., 1992).

With respect to traditional chemical-physical methods, biosorption hasindubitable advantages such as a highly rapid treatment and thepossibility of recovering absorbed dye for future use. Moreover, it canbe carried out also with deactivated biomasses; this has huge advantagesboth thanks to the lower environmental impact and because it is notnecessary to monitor the various factors affecting the growth of aliving organism.

However, there are several factors that might affect biosorption yields,in particular growth substrate, pH, incubation temperature and initialdye concentration (Aksu and Tezer, 2000; Abd El Rahim et al., 2003, AksuZ., 2005).

DESCRIPTION OF THE INVENTION

The invention aims at identifying/selecting fungal species to be used inmethods for treating industrial wastewaters containing dyes.

According to the present invention, such aim is achieved thanks to thesolution specifically disclosed in the following claims. The claims arean integral and substantial part of the technical teaching provided herewith reference to the invention.

In particular, the invention relates to the use of the fungal speciesCunninghamella elegans Lendner in a method for the biosorption ofindustrial dyes, e.g. of the dyeing or tanning industry.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1. Absorbance spectrum of simulated wastewater containing R80 dyeat a concentration of 5,000 ppm, at test beginning (0 h) and after 2, 6and 24 hours of incubation with the biomass of Cunninghamella elegansMUT 2861 pre-grown in a culture medium containing starch as carbonsource (AM).

FIG. 2. Biomass of Cunninghamella elegans MUT 2961 immobilized incalcium alginate and grown in a culture medium containing starch ascarbon source (AM): decrease of mix absorbance and ppm removed from testbeginning and after 6 hours of incubation.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail with reference to apreferred embodiment, provided by way of mere non-limiting example.

In a particular and preferred embodiment of the present invention, thefungal biomass includes the fungal species Cunninghamella elegansLendner in deactivated form, and still more particularly it includes thestrain of Cunninghamella elegans Lendner, MUT 2861, deposited at theDSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Braunschweig, Germany), under access number DSMZ 18657 on Sep. 15, 2006.

The results obtained in the present application show that deactivatedbiomasses of C. elegans Lendner MUT 2861 have high biosorption yields,both towards single dyes belonging to the main classes of industrialdyes (azo and anthraquinone dyes), towards a simulated wastewatercontaining ten dyes differing in chromophore group (azo, anthraquinoneor phthalocyanine group) and in chemical group (acid, reactive or directgroup), and towards three effluent models designed to mime wastesproduced during cotton or wool textile dyeing processes. The added valueof the last result stems from the fact that model effluents wereprepared using mixed commercially important industrial dyes, containhigh concentration of salts and mimic the industrial wastewaters alsofor the pH values introducing real parameters that often bars theattainment of good biosorption yields according to Aksu (2005). Mostworks on biosorption published until today relate to the treatment ofsimulated wastewaters containing single dyes or maximum 2-3 dyessimultaneously, with total concentrations of about 200 ppm and almostnever above 800 ppm (Aksu and Tezer, 2000). The concentrations ofwastewaters used in the present study (up to 5,000 ppm) can therefore beregarded as very high and representative of actual industrialwastewaters.

The comparison with data available from scientific literature shows thatthe values of biosorption capacity for C. elegans MUT 2861 obtainedtowards industrial dyes (279 to 499 mg of dye removed per g of drybiomass) are far higher than values disclosed in the scientificliterature for other living or deactivated and non-deactivated fungalbiomasses (Fu and viraraghavan, 2000; 2002a; O'Mahoney et al., 2002;Zhang et al., 2003; Aksu, 2005), and comparable with theoretical maximumcapacity values for fungus Rhizopus oryzae towards different industrialdyes (Aksu and Tezer, 2000; Aksu and Cagatay, 2006). Table 1 containssorption capacities of living or deactivated biomasses of differentfungal species disclosed in the scientific literature, among which thefungal strain C. elegans MUT 2861 according to the present invention.

TABLE 1 Sorption capacity Fungal species Biomass Dyes used (mg g⁻¹)Authors Aspergillus Deactivated Basic Blue 9 (50 ppm) Up to 18.5 Fu andniger Viraraghavan, 2000 Aspergillus Deactivated Congo Red (50 ppm) Upto 17.6 Fu and niger Basic Blue 9 (50 ppm) Viraraghavan, Acid Blue 29(50 ppm) 2002b Dispers Red 1 (50 ppm) Penicillium Active Reactive Red241 (100 ppm) 115-160 Zhang et al., oxalicum Reactive Blue 19 (100 ppm)2003 Reactive Yellow 145 (100 ppm) Rhizopus Deactivated Reactive Orange16 (250 ppm)  90-190 O'Mahoney et oryzae Reactive Red 4 (250 ppm) al.,2002 Reactive Blue 19 (250 ppm) MIX (450 ppm) Rhizopus DeactivatedReactive Black 5 (800 ppm) Up to 500.7 Aksu and arrhizus = Gemazolturquoise Blue-G Up to 773 Tezer, 2000 R. oryzae (800 ppm) Aksu andCagatay, 2006 Cunninghamella Deactivated Direct Red 80 (5,000 ppm) Up to432.5 * elegans Reactive Blue 214 (5,000 ppm) Up to 427.8 RBBR (5,000ppm) Up to 273.3 Mix of 10 dyes (5,000 ppm) Up to 498.8

C. elegans has never been used before for removing industrial textiledyes as deactivated biomass, which is therefore able to decolourizewastewaters by bioabsorption only. Conversely, the degradationcapacities of living biomasses of C. elegans are well known: as a matterof fact, Cha et al. (2001) have used C. elegans for obtaining thebiotransformation of malachite green into leucomalachite. More recently,Ambrosio and Campos-Takaki (2004) have used this species as livingbiomass for bleaching by biodegradation 3 azo dyes, used individually ata concentration of 0.025 mM, or in combination at a concentration of0.034 mM.

The fast removal of dyes both from simulated wastewaters, and fromeffluent models, as shown in the tests carried out by the inventors ofthe present application, and above all the excellent decolourizationpercentages already obtained after 2 hours of treatment, point out theindustrial applicability of the biomasses of C. elegans MUT 2861.

The results obtained suggest that the chemical structure of dyes canaffect sorption yields. As a matter of fact, azo dyes R80 and B214 havebeen removed from wastewater more easily than anthraquinone dye RBBR.Differences in steric size and/or charge distribution can be the factorsaffecting the interaction between the binding sites on fungus wall anddye molecules. However, the absence of modifications in the absorptionspectra during the trials with the effluent models, showed the capacityof C. elegans MUT 2861 to remove different dyes with the sameefficiency.

In the tests discussed in the present application, biomasses pre-grownin different culture media (EQ or AM) have significantly differentsorption capacities with respect to the same dye.

It is known that the culture medium can modify both the chemicalstructure and the structure of cell wall (Bartniki-Garcia and Nickerson,1962; Farkas, 1980; Krystofova et al., 1998; El-Mougith et al., 1999;Hefnavy et al., 1999; Znidarsic et al, 1999; Nemcovic and Farkas, 2001)as well as colony morphology (Pessoni et al., 2005). According toZnidarsic et al. (1999) the amount and quality of carbon and nitrogensources can affect the amount of structural compounds, such as chitinand chitosane, and of other chemical groups that are present in cellwall.

Highly interesting is the fact that the biomass of C. elegans MUT 2861pre-grown in AM, the culture medium containing starch as carbon source,has shown comparable or higher decolorization percentage and sorptioncapacities towards all the simulated wastewaters and effluent modelstested than the biomass pre-grown in EQ, the culture medium containingglucose. This result is very important from the point of view ofapplication, if the method has to be used on an industrial level; as amatter of fact, starch is a by-product of several industrial processesand represents therefore a low-cost carbon source and the use thereofwould thus enable to reduce biomass production costs, which aregenerally quite high.

The good results obtained with the biosorption test in a column, showthat immobilization in calcium alginate does not affect biosorptionyields. The fungal biomass can be also be used without a supportstructure; for example, it can be introduced directly into theindustrial wastewater to be treated.

Description of Cunninghamella elegans Lendner

Description of fungus C. elegans Lendner grown on Malt Extract Agar at24° C. Wooly colonies, growing very fast and reaching a diameter of 6.6cm in 3 days; first white but tending to take on a dark gray color and apowder-like appearance after the formation of sporangioles.Heterothallic species. Globous, brown Zygospores, diameter of 25-55microns, coated with tuberculate, quite flattened projections.Sporangiophores with a diameter up to 20 microns, with verticillate orsolitary branches; subglobous or pyriform vesicles, end-side with adiameter up to 40 microns, lateral with a diameter of 10-30 microns.Sporangioles with smooth, verrucose or finely echinulate wall, globous(diameter of 7-11 microns) or elliptic shape (9-13×6-10 microns).Optimal growth temperature 25° C., max. 37° C. for some isolates, 50° C.for others

Materials and Methods

The isolate of Cunninghamella elegans Lendner MUT 2861—deposited at DSMZunder access number 18657 on Sep. 15, 2006 and coming from the productmarketed by the same Applicant under trade name Enzyveba Nucleobase—iskept at Mycotheca Universitatis Taurinensis (MUT, Universita di Torino,Dipartimento di Biologia vegetale) as colony in active growth, on AgarMalt medium at a temperature of 4° C. and in freeze-dried formcryopreserved at a temperature of −80° C.

Tested Dyes and Preparation of Simulated Wastewaters and Effluent Models

Simulated Wastewaters

Biosorption tests have been carried out using nine industrial textiledyes (Clariant Italia S.p.a.) and the model dye RBBR (Remazol BrilliantBlue, Sigma-Aldrich, St. Luis, Mo.). The chemical-physical propertiesand, if available, the structural formula of the 10 dyes are listed inTable 2.

For each dye a stock solution at a concentration of 20,000 ppm has beenprepared by dissolving the dye powder in distilled water. Such solutionshave been sterilized by filtration (filters with pores having a diameterof 0.2 μm Schleicher & Schuell GmbH, Dassel, Germany) and stored at 4°C. up to the preparation of the simulated wastewaters.

Since in industrial dyeing processes reactive dyes are released intowastewaters in hydrolyzed form, the stock solutions of dyes B41, B49,B214, R243 and RBBR have been hydrolyzed by means of a 2-hour treatmentat 80° C. with a solution of 0.1 M Na₂CO₃, and then neutralized with asolution of 1N HCl.

The following simulated wastewaters have been used for biosorptiontests:

-   -   saline solution (9 g l⁻¹ NaCl) containing the industrial direct        azo dye R80 at concentrations of 1,000 and 5,000 ppm;    -   saline solution (9 g l⁻¹ NaCl) containing the industrial        reactive azo dye B214 at concentrations of 1,000 and 5,000 ppm.    -   saline solution (9 g l⁻¹ NaCl) containing the anthraquinone type        dye RBBR at concentrations of 1,000 and 5,000 ppm;    -   saline solution (9 g l⁻¹ NaCl) containing all ten dyes at a        final concentration of 5,000 ppm (mix).        Effluent Models

Three effluent models designed to mime wastes produced during cotton orwool dyeing processes were prepared using mixed industrial dyes at highconcentrations. The effluent models were developed by partners of the ECFP6 Project SOPHIED (NMP2-CT-2004-505899) and used under the permissionof the SOPHIED Consortium.

The industrial dyes used in these wastewater models were selectedbecause of representative of the most structural dye types, commerciallyimportant and with a wide range of applications across the textileindustries and were purchased from Town End (Leeds) plc. Thechemical-physical properties and the structural formula of the 10 dyesare listed in Table 3. In addition to the dyes, these effluent modelsmimic the industrial ones also for the presence of different salts,often in high concentrations, and for the pH values.

The first wastewater (R1) contained a mix of 3 acid dyes (300 ppm intotal), and has an ionic strength of 4.23·10⁻² and pH 5. The secondwastewater (R2) contained a mix of 4 reactive dyes previously hydrolyzed(5000 ppm total), and has an ionic strength of 1.26·10⁻¹ and pH 10. Thethird wastewater (R3) contained a mix of 3 direct dyes (3000 ppm total)and has an ionic strength of 1.48 and pH 9. The exact composition of the3 effluent models is listed in table 4. All the mimed effluents weresterilized by tindalization (three 1 hour cycles at 60° C. with 24 hrinterval between cycles at room temperature).

Preparation of Fungal Cultures and Production of Biomass

The reproductive propagules have been taken from colonies in activegrowth aged 7 days, and suspensions have been prepared at a knownconcentration (2.5·10⁵ conidia ml⁻¹) in sterile deionized water using ahemocytometer (Bürker's chamber). One ml of such suspension has beeninoculated into 500 ml flasks containing 250 ml of culture medium. Thefollowing culture media have been used for producing the biomasses:

Culture Medium EQ

glucose 20 g l⁻¹

ammonium tartrate 2 g l⁻¹

KH₂PO₄ 2 g l⁻¹

MgSO₄.7H₂O 0.5 g l⁻¹

CaCl₂.2H₂O 0.1 g l⁻¹

10 ml of a mineral solution containing: 5 mg l⁻¹ MnSO₄.5H₂O, 10 mg l⁻¹NaCl, 1 mg l⁻¹ FeSO₄.7H₂O, 1 mg l⁻¹ CoCl₂.6H₂O, 1 mg l⁻¹ ZnSO₄.7H₂O, 0.1mg l⁻¹ CuSO₄.5H₂O, 0.1 mg l⁻¹ AlK(SO₄)₂, 0.1 mg l⁻¹H₃BO₃, 0.1 mg l⁻¹NaMoO₄.2H₂O.

Culture Medium AM

potato starch 18 g l⁻¹

ammonium tartrate 2 g l⁻¹

KH₂PO₄ 2 g l⁻¹

MgSO₄.7H₂O 0.5 g l⁻¹

CaCl₂.2H₂O 0.1 g l⁻¹

10 ml of a mineral solution containing: 5 mg l⁻¹ MnSO₄.5H₂O, 10 mg l⁻¹NaCl, 1 mg l⁻¹ FeSO₄.7H₂O, 1 mg l⁻¹ CoCl₂.6H₂O, 1 mg l⁻¹ ZnSO₄.7H₂O, 0.1mg l⁻¹ CuSO₄.5H₂O, 0.1 mg l⁻¹ AlK(SO₄)₂, 0.1 mg l⁻¹H₃BO₃, 0.1 mg l⁻¹NaMoO₄.2H₂O.

The use of starch, glucose, sucrose or mixtures thereof is necessary ascarbon source for growing the fungal culture. Such components can beused both as pure substances and as by-products of industrialproductions. For instance, instead of starch potato peels can be used;instead of glucose molasses, bagasse, black liquors deriving fromspirits or sugar cane industry can be used.

Incubation has been carried out under stirring at 110 rpm and at atemperature of 30° C. (thermostatic planetary stirrer Minitron Infors,Bottmingen, CH). After 7-8 days of incubation the biomasses have beentaken from the culture medium by filtration, using a metal sieve withpores having a diameter of 150 μm, and have been rinsed several timeswith saline solution (9 g l⁻¹ NaCl) so as to remove residues of culturemedium that might have interfered with following test stages.

Deactivation of Biomass

The biomasses have been placed in saline solution (9 g l⁻¹ NaCl) anddeactivated by sterilization in autoclave at a temperature of 120° C.for 30 minutes. After such treatment the biomasses have been rinsedseveral times with saline solution.

TABLE 2 Chromo- Common Trade C.I. phore Chemical name name name groupgroup λ_(max) (nm) Chemical structure B113* Nylosan navy blue N-RBL P187 acid blue 113 azo acid 541 →

B214 Drimaren reactive azo reactive 607 navy blue blue X-GN 214 CDG B225Nylosan acid anthra- acid 590-626 blue blue quinone F-2RFL 225 P 160 B41Drimaren reactive phthalo- reactive 616-666 turquoise blue 41 cyanineX-B CDG B49* Drimaren blue P-3RLN GR reactive blue 49 anthra- quinonereactive 586-625 →

B81* Solar blue G P 280 direct blue 81 azo direct 577 →

R111* Nylosan scarlet F-3GL 130 acid red 111 azo acid 499 →

R243 Drimaren reactive azo reactive 517 red X-6BN red 243 CDG R80* Solarred BA P 150 direct red 80 azo direct 540 →

RBBR* Remazol brilliant blue R reactive blue 19 anthra- quinone reactive593 →

Dyes whose chemical structure is shown in the right column.

TABLE 3 Chemical Acronymus C.I. name Chromophore class Chemicalstructure ABk194 Acid black 194 Azoic (1:2 Cr complex) Acid

ABk210 Acid black 210 Trisazoic Acid

AY194 Acid yellow 194 Azoic (1:2 Co complex) Acid

ABu62 Acid blue 62 Anthraquinonic Acid

AR266 Acid red 266 Azoic Acid

AY49 Acid Yellow 49 Monoazoic Acid

DrBu71 Direct blue 71 Trisazoic Direct

DrR80 Direct red 80 Polyazoic Direct

DrY106 Direct Yellow 106 Stilbenic Direct

RBk5 Reactive black 5 Disazoic Reactive

Rbu222 Reactive blue 222 Disazoic Reactive

RR195 Reactive red 195 Monoazoic Reactive

RY145 Reactive Yellow 145 Monoazoic Reactive

TABLE 4 Effluent model Dyes and salts Concentration g l⁻¹ pH Acid bathfor Abu 62 0.10 5 wool AY 49 0.10 (R1) AR 266 0.10 Na₂SO₄ 2.00 Reactivedye Rbu 222 1.25 10 bath for cotton RR195 1.25 (R2) RY145 1.25 Rbk 51.25 Na₂SO₄ 70.00 Direct dye bath DrBu 71 1.00 9 for cotton DrR 80 1.00(R3) DrY 106 1.00 NaCl 5.00Biosorption Tests in a Flask

The biomasses have been divided into 3 g aliquots (fresh weight) andincubated in 50 ml flasks containing 30 ml of simulated wastewater. 3repetitions have been prepared for each test.

Incubation has been carried out under stirring at 110 rpm and at atemperature of 30° C. (thermostatic planetary stirrer Minitron Infors,Bottmingen, CH). After 2, 6 and 24 hours of incubation 300 μl ofsimulated wastewater have been taken for each sample and centrifuged at14,000 rpm for five minutes, so as to remove biomass fragments thatmight have interfered with following spectrophotometric measures.

By means of a spectrophotometer Amersham Biosciences (Fairfield, Conn.),the wastewater absorption spectrum in the visible has been acquired foreach sample (λ=360 nm to λ=790 nm).

In the case of simulated wastewaters, the decolourization percentage(DP), expressed as percentage of removed dye, has been calculated foreach sample according to the following formula:DP=100·[(Abs₀−Abs_(t))/Abs₀]wherein Abs₀ is absorbance at time 0 and Abs_(t) is absorbance at timet, at the maximum wavelength in the visible (λ_(max)) for each dye(Table 2). Mix absorbance has been measured at a wavelength of 588 nm,corresponding to the maximum absorption in the visible.

In the case of effluent models, the DP values were calculated as theextent of decrease of the spectrum area from 360 nm to 790 nm, respectto that of the abiotic control.

Samples of simulated wastewaters and model effluents without biomasshave been used as abiotic controls and for detecting the presence, ifany, of bleaching phenomena not related to biosorption, such asphotodegradation and complexing.

At the end of the test the biomasses have been filtered on filter paper(Whatman type 1), placed in an oven and dried at a temperature of 65° C.for 24 hours, then weighed so as to obtain the dry weight for eachbiomass. It has thus been possible to calculate sorption capacity (SC)according to the following formula:SC=mg of removed dye/g of biomass_((dry weight))

When complete decolourization is achieved, SC is underrated, sinceremoved dye is only part of what the biomass might have removed.

The significance of the differences (p≦0.05) between DP and SC valueshas been calculated by means of Mann-Whitney's non-parametric test(SYSTAT 10 for Windows, SPSS Inc., 2000).

Immobilization of Biomass

For the immobilization of the biomass a conidia suspension has beenprepared as described above, though with such a concentration as toobtain as a result of the mixing with a solution of alginic acid at afinal concentration of 20 g l⁻¹ of alginic acid and of 2.5-104 conidiaml⁻¹. The mixture of alginic acid and conidia, kept under constantstirring by way of a magnetic stirrer, has been dripped by means of aperistaltic pump (model SP311 VELP Scientifica, Milano) into a solutionof 0.25 M calcium chloride, which was also kept under stirring.

Alginic acid hardens immediately in contact with calcium chloride,forming small spheres of calcium alginate with a diameter 2-3 mm, inwhich the propagules are trapped. The small spheres of calcium alginatehave been kept under stirring for about one hour in calcium chloride, soas to obtain a complete hardening thereof, then they are rinsed withsaline solution so as to remove propagules that are not trapped inalginate and the excess of calcium chloride.

In order to obtain the development of the immobilized biomasses, 500 mlflasks have been prepared, each containing 30 g of spheres in 250 ofculture medium AM. The flasks have been incubated under stirring (130rpm) at a temperature of 30° C. (thermostatic planetary stirrer MinitronInfors, Bottmingen, CH). After 7 days of incubation the small spheres ofcalcium alginate have been taken by filtration, using a metal sieve withpores having a diameter of 150 μm, rinsed with physiologic solution anddeactivated by sterilization in autoclave, as described above fornon-immobilized biomasses.

Biosorption Test in a Column with Immobilized Biomass

About 300 g of biomass immobilized in calcium alginate, corresponding to50 g of biomass (fresh weight), net of the weight of calcium alginate,have been packed in a glass column. The column has been connected bymeans of silicone pipes to a flask containing 500 ml of mix simulatedwastewater. Such wastewater has been circulated in the system at aconstant speed of 20 ml min⁻¹ by way of a peristaltic pump (model SP311VELP Scientifica, Milano). After 30 minutes, 1, 2, 3, 4, 5, 6 and 24hours a small amount of wastewater has been taken from the system anddecolourization percentage has been calculated, as described above forbiosorption tests in a flask.

Results

Biosorption Tests in a Flask

Simulated Wastewaters

Decolourization percentages (averages and standard deviations of 3repetitions) of simulated wastewaters containing dyes R80, B214, RBBR ata concentration of 1,000 ppm, after 2, 6 and 24 hours of incubation withbiomasses of Cunninghamella elegans MUT 2861 pre-grown in EQ and AM, areshown in Table 5.

TABLE 5 Culture Decolourization percentage (%) Dye medium 2 hours 6hours 24 hours R80 EQ 83.2 ± 4.9 99.6 ± 0.3 100.0 ± 0.0 AM 73.6 ± 1.998.2 ± 0.4 100.0 ± 0.0 B214 EQ 88.2 ± 1.3 95.3 ± 0.2  99.0 ± 0.1 AM 91.7± 1.4 95.5 ± 0.4  98.8 ± 0.3 RBBR EQ 22.7 ± 3.6 38.0 ± 2.2  57.8 ± 0.9AM 28.8 ± 2.7 46.1 ± 4.1  63.2 ± 3.6

Table 6 shows decolourization percentages (average±standard deviationsof 3 repetitions) of simulated wastewaters containing dyes R80, B214,RBBR and the mix at a concentration of 5,000 ppm, after 2, 6 and 24hours of incubation with biomasses of Cunninghamella elegans MUT 2861pre-grown in EQ and AM.

TABLE 6 Culture Decolourization percentage (%) Dye medium 2 hours 6hours 24 hours R80 EQ 34.2 ± 1.5 43.3 ± 3.9 66.0 ± 2.1 AM 35.6 ± 5.852.5 ± 2.0 78.8 ± 0.5 B214 EQ 53.2 ± 2.4 63.9 ± 1.0 70.2 ± 1.4 AM 53.2 ±2.5 65.1 ± 1.3 71.4 ± 0.4 RBBR EQ 51.5 ± 3.4 49.1 ± 5.7 46.2 ± 9.0 AM52.1 ± 1.1 60.0 ± 2.2 60.6 ± 2.9 Mix EQ 66.4 ± 3.5 79.8 ± 4.3 88.6 ± 1.8AM 69.5 ± 3.6 83.4 ± 2.3 90.7 ± 0.6

With simulated wastewater containing dye R80 at a concentration of 1,000ppm a complete decolourization has been obtained after 24 hours oftreatment with biomasses of C. elegans MUT 2861 pre-grown in bothculture mediums; also with dye B214 high decolourization percentageshave been obtained (98.8% for AM and 99.0% for EQ), whereas RBBR hasproved to be the most difficult dye to remove (57.8% for EQ and 63.2%for AM) (Table 5).

With simulated wastewaters at a concentration of 5,000 ppm, generallygood decolourization percentages have been obtained; in particular withthe mix removal has been above 88.6% (Table 6).

High decolourization yields have been obtained both with wastewaters ata concentration of 1,000 ppm and with wastewaters at 5,000 ppm alreadyafter 2 and 6 hours of treatment, which proves that the biosorptionprocess is rapid and is almost fully completed within few hours.

FIG. 1 shows by way of example absorbance decrease in the absorptionspectrum of the simulated wastewater containing dye R80 at aconcentration of 5,000 ppm, at time zero and after 2, 6 and 24 hours oftreatment, with the biomass of C. elegans MUT 2861 pre-grown in AM.

The monitoring of absorption spectra of simulated wastewaters before andafter treatment shows that decolourization occurs only by means ofbiosorption (no biodegradation takes place), since the spectrum profiledoes not change although dye concentration sinks.

Table 7 shows sorption capacities (average and standard deviations of 3repetitions) of biomasses of C. elegans MUT 2861 pre-grown both in EQand in AM towards simulated wastewaters containing dyes R80, B214, RBBRand the mixture thereof at a concentration of 5,000 ppm.

TABLE 7 Sorption capacity mg of dye g⁻¹ of biomass (average ± standarddeviation) Dye EQ AM R80 278.7 ± 8.7*^(A) 432.5 ± 18.5^(A) B214 327.6 ±7.7*^(B) 427.8 ± 25.2^(A) RBBR  176.1 ± 27.4*^(C) 273.3 ± 16.6^(B) Mix393.1 ± 7.2*^(D) 498.8 ± 2.8^(C)  *refers to significant differences (p≦ 0.05) among values of sorption capacity obtained with the samesimulated wastewater by means of biomasses pre-grown on differentculture media. ^(A,B,C)refer to significant differences (p ≦ 0.05) amongvalues of sorption capacity towards different simulated obtained withwastewaters pre-grown on the same culture medium.

A comparison of the two different culture media used for producing thebiomass (EQ and AM) points out that the sorption capacity of C. elegansMUT 2861 towards all effluents tested is significantly higher when thebiomasses are pre-grown in AM.

RBBR has proved to be the most difficult dye to remove, both withbiomasses pre-grown in EQ and with biomasses pre-grown in AM.

Effluent Models

Table 8 shows decolourization percentages (average±standard deviationsof 3 replicates) of effluent models, after 2, 6 and 24 hours ofincubation with biomasses of C. elegans MUT 2861 pre-grown in EQ and AM.

TABLE 8 Culture Decolourization percentage (%) Effluent medium 2 hours 6hours 24 hours R1 EQ 85.4 ± 3.6^(A) 91.5 ± 0.7^(B) 92.8 ± 1.4^(B) AM83.9 ± 3.1^(A) 88.1 ± 4.1^(A) 93.8 ± 0.4^(B) R2 EQ 50.7 ± 1.8^(A) 56.0 ±3.1^(B) 59.3 ± 3.1^(B) AM 44.7 ± 4.0^(A) 53.2 ± 0.5^(B) 63.8 ± 3.6^(C)R3 EQ 51.8 ± 3.4^(A) 66.5 ± 2.8^(B) 87.5 ± 2.1^(C) AM 57.1 ± 1.5^(A)74.9 ± 3.0^(B) 97.7 ± 0.4^(C) ^(A,B,C)refer to significant differences(p ≦ 0.05) among values of decolourization percentage at differentincubation time obtained by the same biomass.

Substantial decolourization of R1 was achieved with DP values higherthan 93% within 24 hours. In both cases, more than 92% of the DPobtained at the end of the experiment was achieved within 2 hours. Inthe case of the biomass pre-grown in AM the DP significantly increasedfrom 6 to 24 hours.

The DP values of R2 after 24 hours were higher than 59% and most of thetotal decolourization obtained at the end of the experiment (70-85%) wasobtained within 2 hours.

The DP values of R3 after 24 hours were higher than 88%. In comparisonto the other simulated effluents, lower percentage of the totaldecolourization (58-63%) was attained within 2 hours. For both thebiomasses pre-grown on the 2 media significant differences among the DPvalues after 2, 6 and 24 hours were observed.

Biosorption Test in a Column with Immobilized Biomass

The good results obtained with tests in a flask have been confirmed bythe biosorption test in a column, using the immobilized and deactivatedbiomass C. elegans MUT 2861; in this case a complete decolourization ofthe mix at 5,000 ppm after 6 hours of treatment has been obtained (FIG.2).

Obviously, details and embodiments can be widely varied with respect towhat has been here described and shown, although without leaving theprotection scope of the present invention as defined in the appendedclaims.

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1. Use of a fungal biomass for treating industrial wastewaterscontaining at least one dye, wherein: i. the fungal biomass includes atleast the fungal strain Cunninghamella elegans Lendner MUT 2861, DSMZNo. 18657; ii. the fungal biomass absorbs the at least one dye, so as toobtain wastewater that is basically free of the at least one dye.
 2. Theuse according to claim 1, wherein the at least one dye belongs to theclass of azo dyes, anthraquinone dyes or phthalocyanine dyes.
 3. The useaccording to claim 1, wherein the at least one dye contains achromophore group chosen among azo groups, anthraquinone groups,phthalocyanine groups, indigo groups and stilbene groups.
 4. The useaccording to claim 1, wherein the fungal biomass is grown in a culturemedium containing a carbon source.
 5. The use according to claim 4,wherein the culture medium containing a carbon source is selected amongstarch, glucose, sucrose or mixtures thereof.
 6. The use according toclaim 5, wherein the culture medium contains starch.
 7. The useaccording to claim 4, wherein the fungal biomass is grown in a culturemedium further containing an ammonium salt, preferably ammoniumtartrate.
 8. The use according to claim 1, wherein the fungal biomass isdeactivated.
 9. The use according to claim 1, wherein the fungal biomassis immobilized on a support structure.
 10. A method for treatingindustrial wastewaters containing at least one dye, comprising thefollowing steps: a. providing a fungal biomass comprising at least thefungal strain Cunninghamella elegans Lendner MUT 2861, DSMZ 18657; b.contacting the fungal biomass with the industrial wastewater for asufficient lapse of time so as to enable the absorption of the at leastone dye by the fungal biomass, thus obtaining wastewater that isbasically free of the at least one dye.
 11. The method according toclaim 10, wherein that the fungal biomass is immobilized on a supportstructure.
 12. The method according to claim 10, wherein the fungalbiomass is grown in a culture medium containing a carbon source.
 13. Themethod according to claim 12, wherein the culture medium containing acarbon source is selected among starch, glucose, sucrose or mixturesthereof.
 14. The method according to claim 13, wherein the culturemedium contains starch.
 15. The method according to claim 12, whereinthe fungal biomass is grown in a culture medium further containing anammonium salt, preferably ammonium tartrate.
 16. The method according toclaim 12, wherein the fungal biomass is grown in a culture mediumfurther containing at least a salt of K, Mg, Ca, Na, Mn, Fe, Co, Zn, Cu,Al, B and Mo.
 17. The method according to claim 12, wherein the fungalbiomass is separated from the culture medium before step b).
 18. Themethod according to claim 10, wherein the fungal biomass is deactivatedby means of chemical or physical treatment before step b).
 19. Themethod according to claim 18, wherein the fungal biomass is deactivatedby sterilization.